Objective To construct and express recombinant cecropin B-binding site of luteinizing hormone releasing hormone(CB-LHRH')gene,and to evaluate the anticancer function of CB-LHRH' on human ovarian cancer cell line SKOV3 and human endometrial cancer cell line HEC-1B.Methods The sequence of the cDNA encoding CB-LHRH' was designed,artificially synthesized,verified by DNA sequence analysis and expressed by Bac-to-Bac baculovirus expression system.The expression of CB-LHRH' proteins were identified by western dot blot using rabbit polyclonal antibody against LHRH as the primary antibody.To determine the anticancer effects of the CB-LHRH' protein,ovarian cancer cell line SKOV3 and endometrial adenocarcinoma cell line HEC-1B were treated by different doses of the CB-LHRH' protein.Cell growth inhibition assay was performed using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5[(phenylamino) carbonyl]-2H-tetrazolium hydroxide(XTT)kit at different times,and cell morphologic changes were observed under the inverted microscope.Results The inhibitory rate of proliferation by CB-LHRH' increased with the increase of dose and time respectively:SKOV3 cell,from(5.03?0.08)% to(53.24 ?1.22)%;HEC-1B cell,from(5.13?0.37)% to(56.16?1.08)%.The inhibitory effect on HEC-1B cell was stronger than that on SKOV3 cell(P

The genetic analysis of herpesviruses has been a constant challenge, due to the large, complex genomes of herpesviruses and mutagenesis of viral genes by conventional recombination methods in cell culture. Recently, a completely new approach for full-length infectious clones of herpesviruses based on bacterial artificial chromosomes (BACs) has been developed. This technique allows the maintenance, propagation and genetic modification of the viral genome as a BAC plasmid in E.coli, thus making the procedures fast, safe and effective in prokaryotic cells. This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells, thereby facilitating the analysis of viral gene functions in the context of genome. In this presentation, Epstein-Barr virus was used as an example to describe the principle, establishment of the technique and mutation introduction into the BAC plasmid, and to discuss the perspective in the use of BAC-cloned herpesviruses.

Objective To evaluate the clinical efficacy of expulsive therapy using alpha 1-selective adrenoblocker and prostaglandin synthetase inhibitor for distal ureteral stones.Methods 94 patients with distal ureteral stone were randomly divided into study group and control group.In study group,47 cases received tamsulosin 0.2mg daily and dielofenac rectal suppositories 50mg 2 times daily,while watchful waiting without tamsulosin and diclofenac suppository in 47 cases of control group.Observation lasted 2 weeks and also stone expelled as end point.Results No severe adverse reactions related to the drugs were noted and no patients withdrew from the study.The stone-free rate was 91.5%(43/47) of the study group and 25.5% (12/47) of the control group (P＜0.01).A mean stone expelling date of the control group was(8.9±4.3)d and that of the study group was(6.4±3.7)d(P＜0.01).Rates of renoureteral colic recurred in the study group and the control group were 4.3%(2/47) and 48.9%(23/47),respectively (P＜0.01).Conclusion Treatment of distal ureteral stones wlth tamsulosin and dielofenac suppository can increase the stone-free rate,shorten mean stone expelling date,decrease rate of renottreteral colic recurred.

Objective To explore the role of plasminogen activator inhibitor-1(PAI-1)in development of progressive fibrosis via the inhibition of extracellular matrix degradation,and to reveal the contributive role of PAI-1 in muscular dystrophy(MD).Methods Expression and cellular localization of PAI-1 protein were examined in frozen muscle specimens obtained via biopsy from 5 patients with duchenne muscular dystrophy(DMD),3 patients with becker muscular dystrophy(BMD),9 patients with congenital muscular dystrophy(CMD) and 4 cases with normal muscle by immunohistochemistry,double immunofluorescence and Western-blot analysis.Results PAI-1 was positive only in vascular endothelial cells of normal muscle.Both immunohistochemistry and Western-blot analysis showed that PAI-1 expression distinctly increased in most dystrophic muscles of MD than that in normal muscles.Double immunolabeling revealed that PAI-1 strongly expressed in cytoplasm and nuclei of regenerating muscle fibers,macrophages,macrophage infiltrating necrotic fibers.Some activated fibroblasts in endomysium and perimysium of DMD and CMD muscles were positive for PAI-1.Conclusions The functional consequence of overexpression of PAI-1 in dystrophic muscles is unknown but the elevated local expression of PAI-1 in diseased muscles of MD and their distinct distribution pattern provide evidence that PAI-1 participate in pathogenesis of MD.

Objective To explore the high risk factors, pathogenesis and methods of early diagnosis of periventricular leukomalacia(PVL) in premature infants.Methods The history of intrauterine hypoxia-ischemia was investigated in premature infants;TORCH-IgM antibodies in cord blood of premature infants were measured by ELISA; Nitric oxide (NO) levels in their cord blood were determined by nitric acid reducing enzyme means. Results Thirty-nine of 52 premature infants in PVL group had a history of intrauterine hypoxia-ischemia; TORCH-IgM antibody positive rate in cord blood of premature infants in PVL group was significantly higher than that in control group(P

Objective To investigate the clinical feature,image of CT scan pulmonary,diagnosis and treatment response in children with pulmonary cavity,and discuss the method of diagnosis and the tactics of treatment for pulmonary cavity in children.Methods A retrospective study of 6 patients with pulmonary cavity,who were diagnosed and treated from Jul. 2003 to Oct. 2009 in Department of Pediatrics of the First Hospital Affiliated to China Medical University.The clinical manifestations,laboratory tests,image of CT scan pulmonary,microbiological evidence,diagnostic procedure and treatment response were collected and evaluated.Results Six patients all didn′t have history of lung di-sease,there were 4 boys and 2 girls,8-15 years old,average age was 10.5 years old.Two cases of them had unrelated pulmonary underlying diseases,1 case had hyperthyroidism,and the other had juvenile idiopathic arthritis and had complication of macrophage activation syndrome,the other 4 cases had no obvious history.All cases had fever (38-40 ℃),3 cases had cough and 1 case had chest pain.Staphylococcus aureus were cultured in 2 cases,no bacteria was cultured in other 4 cases;the count of white blood cell decreased in 2 cases and increased in 4 cases;C-reactive protein increased in 5 cases and was normal in 1 case;plasma IgE level increased in 2 cases and was normal in other 4 cases;plasma 1,3-beta-D-glucan of all 6 cases were negative.Pulmonary cavities were found in the first CT scan of the lungs in 5 cases and only 1 case of patient′s pulmonary cavities was found in the second CT scan of the lung.Five cases were diagnosed infective causes,1 case was diagnosed noninfectious cause,5 cases of infective causes had been treated with anti-microbial drugs for at least 1 week,1 case of noninfectious cause were treated with methylprednisolone cobined cyclosporin A for 2 weeks.Pulmonary CT scan was rechecked in all cases,and the state of the cases were improved before discharged from hospital.Conclusions The causes of pulmonary cavity in children are not only infective factors,but also some non-infective disease,especially some changes of image of pulmonary CT scan has diagnostic value,detailed past medical history and appropriate rechecking of chest radiographic check are very necessary for diagnosis,according to the result of microbial inspection and evaluation of treatment effect in time and then adjust the treatment protocols.

Objective To detect the changes in serum concentrations of angiogenic factors in patients with unstable angina pectoris.Methods A total of 57 patients with unstable angina pectoris were eligible for study and divided into diabetes group (n =23) and non-diabetes group (n =34).Exclusion criteria included complicated diseases,symptomatic peripheral vascular diseases,renal or liver diseases,cerebral embolism and evidence of ongoing infection.Another 36 age-matched healthy subjects from medical examination center were enrolled as control group.Serum samples were collected,and serum concentrations of vascular endothelial growth factor (VEGF),angiopoietin-1,angiopoietin-2,angiogenin,angiostatin,basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) were measured by using cytokine array technology.The data were analyzed by independent-samples t test with the SPSS 13.0statistic software package.Results Compared with the control group,the serum concentrations of VEGF and angiopoietin-2 were significantly increased in patients with unstable angina pectoris (both P ＜ 0.01).Whereas,no significant differences in serum concentrations of angiogenin,angiopoietin-1,angiostatin,bFGF,and PDGF-BB were detected between control group and patient groups.There were no significant differences in serum concentrations of above all 7 biomarkers between diabetes group and non-diabetes group.Conclusions Serum concentrations of VEGF and angiopoietin-2 were increased in patients with unstable angina pectoris,and diabetes didn' t affect the increases in serum concentrations of VEGF and angiopoietin2 caused by unstable angina pectoris.

Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control (cy3) and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6( IFI6 ) , enhancer of zeste homolog 2 (EZH 2 ) and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.

Objective To study the effects of a miRNA family member,let-7e,on the LPS-induced expression of TNF-α in primary monocytes and the possible mechanism.Methods Peripheral blood mononuclear cells (PBMCs) were isolated form human blood sample by using density gradient centrifugation for further isolation of primary monocytes.Flow cytometry analysis was used to measure the purity of isolated primary monocytes.The efficiencies of transfection were evaluated by qRT-PCR and immunofluorescence assay after transfecting the primary monocytes with let-7e mimic or miRNA mimic negative control (NC) for 24 h,36 h and 48 h.To screen out the optimal stimulation time,ELISA was performed to detect the concentrations of TNF-α in the supernatants of cell culture after stimulating the primary monocytes with 1 mg/L of LPS for 0 h,1 h,3 h,6 h and 12 h,respectively.ELISA and qRT-PCR were used to measure the expression of TNF-α in the transfected cells with the interference of LPS.Western blot assay was used to detect the level of enhancer of zeste homolog 2 (EZH2) in let-7e mimic-transfected primary monocytes and the levels of NF-κB p65,ADP-ribosylation factor GTPase-activating protein 1 (ARFGAP1) and Arfaptin2 in the siEZH2-transfected monocytes.Results More than 70％ of the isolated cells were CD14+ cells.The miRNA mimics could transfect the primary monocytes effectively and the transfection rate was about 70％.High levels of let7e were detected in let-7e mimic-transfected primary monocytes 24 hours after the transfection.High levels of TNF-α were observed in the primary monocytes after stimulated with LPS for 12 h,which was considered as the optimal LPS stimulation time.Results of the ELISA indicated that let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes at both mRNA and protein levels.Western blot assay showed that the levels of EZH2 in the let-7e mimic transfected primary monocytes were significantly lower than that in mimic NC transfected primary monocytes.Silenced expression of EZH2 significantly inhibited the expression of NF-κB p65 in nucleus as well as the expression of ARFGAP1 and Arfaptin2.Conclusion let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes.It is possible that Let-7e regulates the expression of NF-κB p65,ARFGAP1 and Arfaptin2 by targeting EZH2 directly to inhibit the expression of TNF-α.

Objective To investigate the effects of a miRNA family member, let-7e, and a combi-nation of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technol-ogy.Methods The efficiency of transfection was evaluated by immunofluorescence assay after transfecting THP-1 cells with micrONTM mimic negative control (Cy3) for 24 h, 36 h and 48 h.The three miRNA mim-ics (let-7e, miR-106b and miR-20a) were respectively used to transfect the THP-1 cells for 24 h, 36 h and 48 h and the expression of each miRNA was analyzed by qRT-PCR analysis for screening out the optimal transfection time.The transfected THP-1 cells were stimulated with1 mg/L of LPS for 1 h.The Luminex xMAP technology was used to detect the expression of IL-8, interferon-inducible protein-10 (IP-10), mono-cyte chemotactic protein 1 (MCP-1), IL-1α, IL-6, IL-10, TNF-α, IFN-αand IFN-βin the supernatants of cell culture.A statistical analysis was performed to analyze the data obtained by using SPSS16.0 software. Results More than 90% of the transfected THP-1 cells were labeled with red fluorescence.The optimal transfection times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively.Com-pared with the corresponding negative control (NC), the expression of IL-8, IP-10 and MCP-1 by THP-1 cells were enhanced after the transfection with let-7e mimic, but were inhibited after the co-transfection with miR-106b and miR-20a mimics.Conclusion The expression of IL-8, IP-10 and MCP-1 were enhanced in let-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells.